RESUMO
PURPOSE: Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage. METHODS: Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation. RESULTS: Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions. CONCLUSIONS: Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD.
Assuntos
Antioxidantes/uso terapêutico , Suplementos Nutricionais , Luz/efeitos adversos , Lesões Experimentais por Radiação/prevenção & controle , Retina/efeitos da radiação , Degeneração Retiniana/prevenção & controle , Animais , Western Blotting , Sobrevivência Celular , Avaliação Pré-Clínica de Medicamentos , Proteínas do Olho/metabolismo , Masculino , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/etiologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/patologia , Rodopsina/fisiologiaRESUMO
PURPOSE: Zinc oxide effectively reduces visual cell loss in rats exposed to intense visible light and is known to slow the rate of disease progression in advanced stages of age-related macular degeneration. Our goal was to determine the efficacy of zinc oxide in combination with novel and well-established antioxidants in an animal model of light-induced oxidative retinal damage. METHODS: One group of male Sprague-Dawley rats was pretreated with zinc oxide with or without a detergent extract of rosemary powder and then exposed to intense visible light for 4-24 h. Another group of animals received zinc oxide combined with rosemary oil diluted with a mixture of polyunsaturated fatty acids (ROPUFA) and a third group was given an antioxidant mineral mix containing zinc oxide, as recommended by the Age Related Eye Disease Study group's first clinical trial (AREDS1). Visual cell survival was determined 2 weeks after intense light treatment by measuring rhodopsin and photoreceptor cell DNA levels and confirmed by retinal histology and agarose gel electrophoresis of DNA. Western analysis was used to determine the effects of zinc and antioxidants on the oxidative stress markers, glial fibrillary acidic protein (GFAP), heme-oxygenase-1 (HO-1), and carboxyethylpyrrole (CEP). Rod and cone opsin and arrestin levels were used as markers of photoreceptor cell function. RESULTS: Dark-reared rats treated with 1.3 mg/kg zinc oxide and 17 mg/kg rosemary extract, or with one-half those doses, and exposed to moderate intensity green light retained 75%-85% of the rhodopsin and retinal DNA measured in unexposed rats. These levels were significantly higher than found for zinc oxide or rosemary treatment alone. Rosemary oil was also effective when combined with zinc oxide, but ROPUFA alone was no more effective than the detergent vehicle. Prolonged intense green light led to increases in retinal GFAP and HO-1 levels and to decreases in cone cell opsin and rod and cone arrestins. Rosemary plus zinc treatment reduced the expression of oxidative stress protein markers and enhanced visual cell survival, as shown by improved photoreceptor cell morphology and by decreased retinal DNA degradation. Using higher intensity white light for exposures in cyclic light-reared rats, treatment with an AREDS antioxidant/mineral mixture was found to be ineffective, whereas rosemary extract plus an equivalent dose of zinc oxide was significantly more effective in preserving visual cells. CEP protein adduct formation was reduced by all antioxidant treatments, but rosemary plus zinc oxide also prevented the loss of cone cell opsin and arrestin more effectively than AREDS. CONCLUSIONS: In the rat model of acute retinal light damage, zinc oxide combined with a detergent extract of rosemary powder or rosemary oil is more effective than treatment with either component alone and significantly more effective than an AREDS mixture containing a comparable dose of zinc oxide. Light-induced oxidative stress in animal models of retinal degeneration can be a useful preclinical paradigm for screening novel antioxidants and for testing potential therapeutics designed to slow the progression of age-related ocular disease.
Assuntos
Extratos Vegetais/farmacologia , Retina/efeitos dos fármacos , Retina/patologia , Rosmarinus/química , Óxido de Zinco/farmacologia , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Eletroforese em Gel de Poliacrilamida , Ácidos Graxos Insaturados/farmacologia , Masculino , Óleos Voláteis/farmacologia , Substâncias Protetoras/farmacologia , Ratos , Ratos Sprague-Dawley , Retina/efeitos da radiaçãoRESUMO
The damaging effects of intense light on the rat retina are known to vary depending on the time of day of exposure. The purpose of this study was to determine if rhodopsin phosphorylation patterns, a measure of the activity of the pigment, varied in a similar manner. After 10 min in strong light (1400 lux), all six threonine and serine sites in the rat rhodopsin C-terminus were phosphorylated, with mono- to tetraphosphorylation being substantially more prominent than penta- to hexaphosphorylation. The level and multiplicity of rhodopsin phosphorylations were reduced both with the duration of light exposure and the duration of subsequent darkness. Although showing vast differences in susceptibility to light damage, rats exposed at 5 P.M. or 1 A.M. showed similar rhodopsin phosphorylation levels and patterns. These data indicate that a process controlled by circadian rhythm other than rhodopsin phosphorylation is involved either in damaging or mediating the damage evoked by intense light exposure.
Assuntos
Ritmo Circadiano/efeitos da radiação , Luz/efeitos adversos , Epitélio Pigmentado Ocular/metabolismo , Lesões Experimentais por Radiação/metabolismo , Rodopsina/metabolismo , Animais , Adaptação à Escuridão , Espectrometria de Massas , Fosforilação , Ratos , Rodopsina/químicaRESUMO
The protective effect of dimethylthiourea (DMTU) against retinal light damage was determined in albino rats reared in darkness or in weak cyclic light. Rats maintained under these conditions were treated with DMTU at different concentrations and dosing schedules and then exposed for various times to intense visible light, either intermittently (1 hr light and 2 hr dark) or continuously. The extent of retinal light damage was determined 2 weeks after light exposure by comparing rhodopsin levels in experimental rats with those in unexposed control animals. To determine the effect of DMTU on rod outer segment (ROS) membrane fatty acids, ROS were isolated immediately after intermittent light exposure, and fatty acid compositions were measured. The time course for DMTU uptake and its distribution in serum, retina, and the retinal pigment epithelium (RPE)/choroid complex was determined in other rats not exposed to intense light. After intraperitoneal injection of the drug (500 mg/kg body weight), DMTU appeared rapidly in the serum, retina, and the RPE and choroid. In the ocular tissues, it was distributed 70-80% in the retina and 20-30% in the RPE and choroid. This antioxidant appears to have a long half-life because it was present in these same tissues 72 hr after a second intraperitoneal injection. For rats reared in the weak cyclic light environment, DMTU (two injections) provided complete protection against rhodopsin loss after intense light exposures of up to 16 hr. Only 15% rhodopsin loss was found in cyclic-light DMTU-treated rats after 24 hr of intermittent or continuous light. For rats reared in darkness, DMTU treatment resulted in a rhodopsin loss of less than 20% after 8-16 hr of continuous light and approximately 40% after similar exposure to intermittent light. Irrespective of the type of light exposure, rhodopsin loss in the dark-reared DMTU-treated rats was nearly identical to that found in uninjected cyclic light-reared animals. In rats from both light-rearing environments, DMTU treatment prevented the light-induced loss of docosahexaenoic acid from ROS membranes. As measured by rhodopsin levels 2 weeks later, DMTU was most effective when given as two doses administered 24 hr before and just before intense light exposure. As a single dose given during continuous light exposure, DMTU protected cyclic light-reared rats for at least 4 hr after the start of exposure but was ineffective in dark-reared animals if injected 1 hr after the start of light. It was also ineffective in both types of rats when given after light exposure.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Luz/efeitos adversos , Doenças Retinianas/prevenção & controle , Tioureia/análogos & derivados , Animais , Corioide/metabolismo , Ritmo Circadiano , Adaptação à Escuridão , Ácidos Graxos/metabolismo , Masculino , Epitélio Pigmentado Ocular/metabolismo , Ratos , Ratos Endogâmicos , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/efeitos da radiação , Doenças Retinianas/metabolismo , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/patologia , Tioureia/farmacocinética , Tioureia/uso terapêuticoRESUMO
PURPOSE: To determine the relative susceptibility of rats to retinal light damage at different times of the day or night. METHODS: Rats maintained in a dim cyclic light or dark environment were exposed to a single dose of intense green light beginning at various times. Normally, light exposures were for 8 or 3 hours, respectively, although longer and shorter periods were also used. Some animals were treated with the synthetic antioxidant dimethylthiourea (DMTU) before or after the onset of light. The extent of visual cell loss was estimated from measurements of rhodopsin and retinal DNA levels 2 weeks after light treatment. The time course of retinal DNA fragmentation, and the expression profiles of heme oxygenase-1 (HO-1) and interphotoreceptor retinol binding protein (IRBP) were determined 1 to 2 days after exposure. RESULTS: When dark-adapted, cyclic light-reared or dark-reared rats were exposed to intense light during normal nighttime hours (2000-0800) the loss of rhodopsin or photoreceptor cell DNA was approximately twofold greater than that found in rats exposed to light during the day (0800-2000). The relative degree of light damage susceptibility persisted in cyclic light-reared rats after dark adaptation for up to 3 additional days. For rats reared in a reversed light cycle, the light-induced loss of rhodopsin was also reversed. Longer duration light treatments revealed that dim cyclic light-reared rats were three- to fourfold more susceptible to light damage at 0100 than at 1700 and that dark-reared animals were approximately twofold more susceptible. Intense light exposure at 0100 resulted in greater retinal DNA fragmentation and the earlier appearance of apoptotic DNA ladders than at 1700. The extent of retinal DNA damage also correlated with an induction of retinal HO-1 mRNA and with a reduction in IRBP transcription. Antioxidant treatment with DMTU was effective in preventing retinal light damage when given before but not after the onset of light. CONCLUSIONS: These results confirm earlier work showing greater retinal light damage in rats exposed at night rather than during the day and extend those findings by demonstrating that a single, relatively short, intense light exposure causes a circadian-dependent, oxidatively induced loss of photoreceptor cells. The light-induced loss of photoreceptor cells is preceded by DNA fragmentation and by alterations in the normal transcriptional events in the retina and within the photoreceptors. The expression profile of an intrinsic retinal factor(s) at the onset of light exposure appears to be important in determining light damage susceptibility.
Assuntos
Ritmo Circadiano/efeitos da radiação , Luz/efeitos adversos , Lesões Experimentais por Radiação/etiologia , Retina/efeitos da radiação , Degeneração Retiniana/etiologia , Tioureia/análogos & derivados , Animais , Northern Blotting , Dano ao DNA/efeitos da radiação , Fragmentação do DNA/efeitos da radiação , Adaptação à Escuridão , Eletroforese em Gel de Ágar , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Sequestradores de Radicais Livres/farmacologia , Perfilação da Expressão Gênica , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Masculino , Lesões Experimentais por Radiação/metabolismo , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Degeneração Retiniana/metabolismo , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Rodopsina/metabolismo , Tioureia/farmacologiaRESUMO
PURPOSE: To compare retinal light damage in rats with either normal or reduced levels of rod outer segment (ROS) docosahexaenoic acid. METHODS: Weanling male albino rats were maintained in a weak cyclic light environment and fed either a nonpurified control diet or a purified diet deficient in the linolenic acid precursor of docosahexaenoic acid (DHA). Half the rats on the deficient diet were given linseed oil, containing more than 50 mol% linolenic acid, once a week to maintain ROS DHA at near normal levels. Diets and linseed oil supplementation were continued for 7 to 12 weeks. To replenish DHA in their ROS, some 10-week-old rats on the deficient diet were given linseed oil three times a week for up to 3 additional weeks. Groups of animals were killed at various times for ROS fatty acid determinations or were exposed to intense green light using intermittent or hyperthermic light treatments. The extent of retinal light damage was determined biochemically by rhodopsin or photoreceptor cell DNA measurements 2 weeks after exposure and morphologically by light and electron microscopy at various times after light treatment. RESULTS: Rats maintained for 7 to 12 weeks on the linolenic acid-deficient diet had significantly lower levels of DHA and significantly higher levels of n-6 docosapentaenoic acid (22:5n-6) in their ROS than deficient-diet animals supplemented once a week with linseed oil or those fed the nonpurified control diet. As determined by rhodopsin levels and photoreceptor cell DNA measurements, deficient diet rats exhibited protection against retinal damage from either intermittent or hyperthermic light exposure. However, the unsaturated fatty acid content of ROS from all three dietary groups was the same and greater than 60 mol%. In 10 week-old deficient-diet rats given linseed oil three times a week, ROS DHA was unchanged for the first 10 days, whereas 22:5n-6 levels declined by 50%. After 3 weeks of treatment with linseed oil, ROS DHA and 22:5n-6 were nearly the same as in rats supplemented with linseed oil from weaning. The time course of susceptibility to retinal light damage, however, was different. Hyperthermic light damage in rats given linseed oil for only 2 days was the same as for rats always fed the deficient diet. Six days after the start of linseed oil treatment, retinal light damage was the same as in rats given the linseed oil supplement from weaning. Morphologic alterations in ROS of linseed oil-supplemented rats immediately after intermittent light exposure were more extensive than in either the deficient-diet animals or those fed the control diet. The deficient-diet rats also exhibited better preservation of photoreceptor cell nuclei and structure 2 weeks after exposure. CONCLUSIONS: Rats fed a diet deficient in the linolenic acid precursor of DHA are protected against experimental retinal light damage. The relationship between retinal light damage and ROS lipids does not depend on the total unsaturated fatty acid content of ROS; the damage appears to be related to the relative levels of DHA and 22:5n-6.
Assuntos
Ácidos Docosa-Hexaenoicos/metabolismo , Luz/efeitos adversos , Lesões Experimentais por Radiação/prevenção & controle , Retina/efeitos da radiação , Segmento Externo da Célula Bastonete/metabolismo , Animais , DNA/análise , Gorduras na Dieta/administração & dosagem , Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos/metabolismo , Masculino , Células Fotorreceptoras/patologia , Células Fotorreceptoras/efeitos da radiação , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/efeitos da radiação , Ácido alfa-Linolênico/deficiênciaRESUMO
PURPOSE: To study the time course of visual cell damage resulting from hyperthermic light exposure and the possible involvement of rod outer segment (ROS) lipids in the process. METHODS: Rats were acclimated in darkness for 2 hours in a hyperthermic chamber to elevate core body temperature and then exposed to intense green light for up to 4 hours during hyperthermia. After light exposure, the animals were either sacrificed immediately for biochemical or morphologic analysis of retinal light damage or returned to darkness for up to 2 weeks at ambient temperature before analysis. Rod outer segment lipid profiles were characterized, and visual cell loss was determined by rhodopsin and visual cell DNA measurements. Morphology was performed at the light and electron microscopic level. RESULTS: Retinal damage resulting from hyperthermic light exposure was found to be temperature, time, and light intensity dependent. At an elevated environmental temperature of 34.5 degrees, 50% visual cell loss was found after 1.5 hours of 1100 lux light exposure; the same degree of visual cell loss occurred after only 1 hour when rats were maintained at 37 degrees C. At ambient temperatures, 4 hours of light exposure had no effect on visual cell loss. Irrespective of environmental temperature, when rats were maintained in darkness no visual cell loss occurred. Whereas docosahexaenoic acid (22:6) was unchanged in the purest fraction of ROS isolated immediately after light treatment, a 5 mol% loss of the polyunsaturated fatty acid was found in ROS isolated 2 or 24 hours after light exposure. Rod outer segment lipid composition was largely unaffected by hyperthermic light exposure, but the density of some ROS increased. Morphologically, the ROS appeared to be nearly normal immediately after hyperthermic light exposure and structurally more abnormal 2 and 24 hours later. The retinal pigment epithelium exhibited damage immediately after exposure, which also increased 2 and 24 hours later. CONCLUSIONS: Hyperthermia in rats dramatically accelerates retinal light damage compared with light exposure under euthermic conditions. Over loss of ROS 22:6 does not occur during hyperthermic light exposure, but it is apparent during the 24-hour period after light treatment. This suggests that the disappearance of 22:6 from ROS occurs in tandem with the process of visual cell death resulting from retinal light damage.
Assuntos
Hipertermia Induzida/efeitos adversos , Luz/efeitos adversos , Lesões Experimentais por Radiação/etiologia , Retina/efeitos da radiação , Animais , DNA/metabolismo , Adaptação à Escuridão , Temperatura Alta , Metabolismo dos Lipídeos , Masculino , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Sprague-Dawley , Retina/metabolismo , Retina/patologia , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Fatores de TempoRESUMO
PURPOSE: To determine the effects of age and long-term light- or dark-rearing environments on acute, intense-light-mediated retinal degeneration. METHODS: Male albino rats were maintained in a dim cyclic light environment or in darkness for as long as 1 year. When aged 2, 4, 8, and 12 months, some rats were given the synthetic antioxidant dimethylthiourea (DMTU) by intraperitoneal injection and were exposed to intense visible light for as long as 24 hours. Uninjected control rats were exposed to light at the same time. Other rats were treated with light of lower intensity for various periods. Two weeks after intense-light treatment, photoreceptor cell degeneration was estimated by determining the level of rhodopsin and by measuring the content of photoreceptor cell DNA. Light-induced changes in retinal DNA were analyzed immediately after exposure by neutral gel electrophoresis and by 8-hydroxy-deoxyguanosine measurements. Expression of the antioxidative stress protein heme oxygenase-1 (HO-1) was determined by northern blot analysis of mRNA in retinal extracts. RESULTS: At all ages, rats reared in cyclic dim-light conditions had lower rhodopsin levels than did rats reared in darkness; photoreceptor cell DNA levels were unaffected by the rearing environment. Senescent losses in rhodopsin and retinal DNA were significant after rats were 12 months old. Dim-light-reared rats exhibited an age-related increase in retinal light damage susceptibility, whereas dark-reared rats were equally susceptible to damage at all ages. In both types of rats, the mechanism of light-induced cell death involved an apoptotic process, visualized by the pattern of DNA fragments on electrophoretic gels. The process also induced the expression of HO-1 mRNA. Photoreceptor cell loss determined by biochemical measurement, DNA fragmentation, and HO-1 induction were dramatically reduced by the administration of DMTU. CONCLUSIONS: The age-related increase in susceptibility to retinal light damage in rats is influenced by their long-term daily light history. Decreasing retinal irradiance by dark-rearing eliminates the age-related increase in light damage, suggesting a correlation between light environment and retinal gene expression associated with damage. In all rats, retinal light damage resulted in a pattern of DNA fragmentation consistent with apoptotic cell death and in an increased expression of HO-1 mRNA. Antioxidant treatment greatly reduced apoptosis and HO-1 expression. This indicates that light damage involves an oxidative process that may also trigger apoptosis in the retina. The rat aging model may provide useful insights into the role of light environment associated with retinal degeneration in an aging human population.
Assuntos
Envelhecimento , Luz/efeitos adversos , Lesões Experimentais por Radiação/metabolismo , Retina/efeitos da radiação , Degeneração Retiniana/metabolismo , 8-Hidroxi-2'-Desoxiguanosina , Envelhecimento/fisiologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/efeitos da radiação , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , Adaptação à Escuridão , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Eletroforese em Gel de Poliacrilamida , Sequestradores de Radicais Livres/farmacologia , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Masculino , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/prevenção & controle , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/metabolismo , Degeneração Retiniana/etiologia , Degeneração Retiniana/prevenção & controle , Rodopsina/metabolismo , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
The damaging effects of visible light on the mammalian retina can be detected as functional, morphological or biochemical changes in the photoreceptor cells. Although previous studies have implicated short-lived reactive oxygen species in these processes, the termination of light exposure does not prevent continuing damage. To investigate the degenerative processes persisting during darkness following light treatment, rats were exposed to 24 h of intense visible light and the accumulation of DNA damage to restriction fragments containing opsin, insulin 1 or interleukin-6 genes was measured as single-strand breaks (ssb) on alkaline agarose gels. With longer dark treatments all three DNA fragments showed increasing DNA damage. Treatment of rats with the synthetic antioxidant dimethylthiourea prior to light exposure reduced the initial development of alkali-sensitive strand breaks and allowed significant repair of all three DNA fragments. The time course of double-strand DNA breaks was also examined in specific genes and repetitive DNA. Nucleosomal DNA laddering was evident immediately following the 24 h light treatment and increased during the subsequent dark period. The increase in the intensity of the DNA ladder pattern suggests a continuation of enzymatically mediated apoptotic processes triggered during light exposure. The protective effects of antioxidant suggests that the light-induced DNA degradative process includes both early oxidative reactions and enzymatic processes that continue after cessation of light exposure.
Assuntos
Dano ao DNA , DNA/efeitos da radiação , Escuridão , Luz , Retina/efeitos da radiação , Animais , Antioxidantes/farmacologia , DNA/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/metabolismo , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
Intense visible light can damage retinal photoreceptor cells by photochemical or thermal processes, leading to cell death. The precise mechanism of light-induced damage is unknown; however, oxidative stress is thought to be involved, based on the protective effect of antioxidants on the light-exposed retina. To explore the in vivo effects of light on retinal DNA, rats were exposed to intense visible light for up to 24 h and the time courses of single-strand breaks in restriction fragments containing the opsin, insulin 1 and interleukin-6 genes were measured. All three gene fragments displayed increasing single-strand modifications with increasing light exposure. Treatment with the antioxidant dimethylthiourea prior to light exposure delayed the development of net damage. The time course of double-strand DNA damage was also examined in specific genes and in repetitive DNA. The appearance of discrete 140-200 base-pair DNA fragments after 20 h of light exposure implicated a nonrandom, possibly enzymatic damaging mechanism. The generation of nucleosome core-sized DNA fragments, in conjunction with single-strand breaks, suggests two phases of light-induced retinal damage, with random attack on DNA by activated oxygen species preceding enzymatic degradation.
Assuntos
Dano ao DNA , Retina/efeitos da radiação , Animais , DNA/genética , DNA/efeitos da radiação , Luz , Masculino , Fotobiologia , Ratos , Ratos Sprague-Dawley , Retina/químicaRESUMO
In the rat, photoreceptor cell death from exposure to intense visible light can be prevented by prior treatment with antioxidants. In this study we subjected albino rats raised in dim cyclic light and rats made more susceptible to light damage by rearing in darkness to exposures of green light that led to similar losses of photoreceptor cells. Rhodopsin and photoreceptor DNA, indicators of the number of surviving photoreceptor cells, were determined at various times over a period of 14 days after light exposure. Fragmentation of DNA was determined over a similar time course by neutral and alkaline agarose gel electrophoresis. Apoptosis in retinal DNA was measured by quantitating the appearance of 180 base pair (bp) nucleosomal fragments. Oxidation of DNA was measured by electrochemical detection of the nucleoside 8-hydroxydeoxyguanosine (8-OHdG) after separation by high-performance chromatography. For albino rats reared in dim cyclic light, 24 h of intense light exposure resulted in the loss of 50% rhodopsin and photoreceptor cell DNA. In dark-reared rats, the losses were 40%, respectively, after only 3 h of intense light treatment. In both cases pretreatment with the antioxidant dimethylthiourea (DMTU) prevented rhodopsin and photoreceptor cell DNA loss. The kinetics of the light-induced apoptosis depended markedly on the rearing environment of the rats. The DNA ladders appeared within 12 h of the onset of intense light in the rats reared in dim cyclic light. In these rats the 180 bp fragment was at two-thirds of its maximum intensity immediately after 24 h of light exposure and reached the maximum 12 h later. Dimethylthiourea partially inhibited ladder formation in rats reared in dim cyclic light and delayed the time of appearance of the 180 bp maximum by 6 h. By contrast, in rats reared in darkness the 180 bp fragment was undetected immediately after 3 h of light exposure and reached its maximum 2 days later. Pretreatment with DMTU completely eliminated DNA ladders in these rats. Alkaline gel electrophoresis revealed a pattern of single-strand DNA breaks, with relatively high molecular weight fragments, 6 h after light exposure of dark-reared rats. Single-strand DNA breaks in cyclic light rats corresponded with the onset of apoptotic ladders, but peak values preceded by 12 h the peak of DNA ladder formation. The quantity of 8-OHdG in retinal DNA remained close to control values in all samples with the exception of a peak of twice the control value 18 h after light exposure in the dark-reared rats and a value 60% higher 16 days after exposure in cyclic light animals. Dimethylthiourea had no effect on the amount of oxidized purine in any of the samples. The differences between dark-reared rats and rats reared in dim cyclic light in the kinetics of DNA fragmentation and in their response to treatment with DMTU is consistent with previous observations of fundamental differences in retinal cell physiology in these animals. In dim light-reared rats, the pathway to apoptosis may be qualitatively different from the pathway to net photoreceptor loss in rats reared in darkness. The lack of effect of DMTU on 8-OHdG formation suggests that the oxidation of DNA bases is not a causal factor in light-mediated photoreceptor cell death.
Assuntos
Retina/lesões , Retina/efeitos da radiação , Animais , Antioxidantes/farmacologia , Apoptose , Sobrevivência Celular , Fragmentação do DNA , Luz , Masculino , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismo , Células Fotorreceptoras/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Tioureia/análogos & derivados , Tioureia/farmacologiaRESUMO
To assess the protective effect of ascorbic acid in retinal light damage of rats, we have determined the uptake and retinal tissue distributions of its L- and D- stereoisomers following interperitoneal or intraocular injections. The effects of intense-intermittent light exposure and darkness on tissue ascorbate were compared by measuring its levels in retina and retinal pigment epithelial tissues at various times after administration. The protective effects of the two forms of ascorbate against retinal light damage were also compared by measuring rhodopsin levels 2 weeks after intense light exposure. After interperitoneal injection, both forms of ascorbic acid were higher in the retinal pigment epithelial-choroid-scleral complex (eye cup) than in the retina. Over a 2 hr post-injection period, L-ascorbate in the eye cup was 2 to 4 fold higher than normal (10-11 nmol); D-ascorbate levels were between 15 and 30 nmol. During the same period retinal L-ascorbate was just above normal (12-14 nmol), whereas less than 5 nmol of D-ascorbate was present. When ascorbate was given by the intraocular route the opposite effect was found. During the 2 hr post-injection period retinal L-ascorbate levels were 2 to 5 fold higher than normal; D-ascorbate was between 25 and 50 nmol/retina. Within 1 hr post-injection, L-ascorbate in the eye cup was near normal and D-ascorbate levels were 10 nmol or less. In uninjected rats perfused with normal saline, the endogenous L-ascorbate was distributed 55% in the retina with 9% and 36%, respectively, in the RPE-choroid and sclera. Ten-thirty min after interperitoneal peritoneal injection about 40% of the L-ascorbate was present in the retina with 17% and 44% in the RPE-choroid and sclera. Total ascorbate (L + D) levels in the same tissues of D- injected rats were similar to those found for rats given L-ascorbate. Following 7 hrs of darkness, tissue ascorbate levels in the injected rats decreased to approximately the same levels present in uninjected animals. For rats exposed to intense light average retinal ascorbate levels decreased further, while RPE-choroid and scleral levels were largely unchanged from the dark control levels. About 50% of the tissue ascorbate was present in the retina 10-30 min after intraocular injection. The RPE-choroid contained between 10 and 14% of the ascorbate, with 35-40% present in the sclera. Retinal ascorbate levels remained high in the injected eyes following 2.5 hrs of darkness, but decreased as a result of intense light treatment.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Ácido Ascórbico/farmacocinética , Luz/efeitos adversos , Retina/metabolismo , Animais , Ácido Ascórbico/administração & dosagem , Cromatografia Líquida de Alta Pressão , Adaptação à Escuridão , Injeções , Injeções Intraperitoneais , Masculino , Ratos , Ratos Endogâmicos , Retina/efeitos da radiação , Degeneração Retiniana/metabolismo , Degeneração Retiniana/prevenção & controle , Rodopsina/metabolismo , Estereoisomerismo , Fatores de Tempo , Distribuição TecidualRESUMO
PURPOSE: To determine the effect of acute, intense, visible light on the activities of all-trans retinol dehydrogenase (t-RDH) and glutamine synthetase (GS), two oxidatively sensitive enzymes located in the photoreceptors and Müller cells, respectively. METHODS: Male albino rats, previously maintained in a weak cyclic light- or dark-rearing environment, were exposed to intense light (490-580 nm) for as long as 24 hrs. One-half of the experimental animals were pre-treated with the antioxidant 1,3-dimethylthiourea (DMTU), at 500 mg/kg, i.p., 24 hrs before and just before light exposure. Upon sacrifice, retinas were excised for the determination of t-RDH and GS activity, or for the preparation of rod outer segments (ROS). Other light-exposed rats were maintained in darkness for 2 weeks before sacrifice, for rhodopsin determinations. Retinal homogenates were also treated in vitro under oxidizing conditions to compare enzymatic inactivation with the in vivo effects of light exposure. RESULTS: In cyclic light-reared rats 24 hr light exposures resulted in a significant loss of t-RDH activity in retinal homogenates and in isolated ROS. In both the retina and ROS, pretreatment of the animals with DMTU completely prevented the loss of t-RDH activity. As measured by rhodopsin levels 2 weeks after light exposure, DMTU-treated rats exhibited no loss of photoreceptor cells, whereas those not given the antioxidant lost over 50% of their photoreceptors. Retinal GS activity was unchanged by 24 hr intense light exposures. In dark-reared rats 4 hr light exposures did not alter retinal t-RDH or GS activity, despite the loss or approximately 70% of the rhodopsin content or the eye, measured 2 weeks later. When 4 hr light-exposed rats were held in darkness for an additional 20 hrs, a significant loss of retinal t-RDH occurred, but no change in GS activity was measured. In these rats DMTU treatment also prevented the loss of t-RDH activity. In contrast to the lack of an in vivo light effect on retinal GS, oxidation in vitro completely inactivated the enzyme after only 1 hr. CONCLUSIONS: The light-induced loss of t-RDH in both cyclic light- and dark-reared rats is an oxidative and time dependent process that is not strictly photochemical in nature. The loss of rhodopsin and t-RDH activity, but not GS activity, following intense light exposure are manifestations of light's effect on photoreceptor cells without a comparable effect in the adjacent retinal Müller cells. Additional work will be needed to understand the differences in light damage susceptibility between retinal photoreceptors and glial cells and between cyclic light- and dark-reared rats.
Assuntos
Oxirredutases do Álcool/metabolismo , Luz , Retina/enzimologia , Retina/efeitos da radiação , Segmento Externo da Célula Bastonete/efeitos da radiação , Animais , Antioxidantes/farmacologia , Família 2 do Citocromo P450 , Adaptação à Escuridão/fisiologia , Glutamato-Amônia Ligase/metabolismo , Masculino , Oxirredução , Fotoperíodo , Ratos , Ratos Sprague-Dawley , Retina/citologia , Segmento Externo da Célula Bastonete/efeitos dos fármacos , Estereoisomerismo , Frações Subcelulares/enzimologiaRESUMO
PURPOSE: To determine the effects of genetic background and light rearing conditions on intense-light-mediated retinal degeneration in young RCS rats. MATERIALS AND METHODS: Albino rats, homozygous or heterozygous for the rdy gene were bred and born in dim cyclic light. At P7 they were moved to a dark environment, and maintained there until exposure to intense visible (green) light at P18 or P25. Other rats remained in the dim cyclic light environment. At various times between P11 and P40 rats were killed for determinations of rhodopsin and photoreceptor cell DNA levels, western transblot analysis of retinal S-antigen (arrestin) and alpha-transducin, or northern slot blot analysis of their respective mRNA levels. RESULTS: At P18, unexposed dark maintained homozygous RCS rats and their phenotypically normal heterozygous counterparts have nearly equivalent rhodopsin levels and photoreceptor cell DNA. Intense light exposure at this age, to 8 hours of continuous light or 3 hours of intermittent light, did not lead to a loss of either rhodopsin or retinal DNA when compared with their respective unexposed controls. At P25 rhodopsin levels were higher than at P18, while photoreceptor cell DNA was essentially the same as in the younger rats. However, intense light exposure at P25 resulted in substantial losses of rhodopsin and photorecptor cell DNA and the losses were greater in homozygous rats than in heterozygous animals. Light damage of P25 rats maintained in dim cyclic light was essentially the same as in dark maintained homozygous rats, but no damage was found in the heterozygous animals. By western analysis, alpha-transducin levels in the retina increased with time in darkness, while retinal S-antigen levels either remained the same or decreased during the period P15-P35. For rats in the cyclic light environment S-antigen expression was greater than alpha-transducin at all ages. Slot blot analysis of mRNAs for the two proteins generally followed the patterns seen by western analysis. S-antigen mRNA was expressed at an earlier age and at higher levels than alpha-transducin in both types of rats from both light rearing conditions. Peak expression of S-antigen most often occurred at P18 in both the heterozygous and homozygous rats. CONCLUSIONS: The relative expressions of S-antigen and alpha-transducin in P18 and P25 rats correlates with their relative resistance to retinal light damage at P18 and their enhanced susceptibility at P25. Rats homozygous for the rdy gene also exhibit more damage than heterozygous animals when photoreceptor cell DNA is used to estimate the extent of retinal light damage.
Assuntos
Células Fotorreceptoras de Vertebrados/patologia , Células Fotorreceptoras de Vertebrados/efeitos da radiação , Lesões Experimentais por Radiação , Ratos Endogâmicos/fisiologia , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Envelhecimento/metabolismo , Animais , Animais Recém-Nascidos/crescimento & desenvolvimento , Animais Recém-Nascidos/metabolismo , Arrestina/genética , DNA/metabolismo , Heterozigoto , Homozigoto , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/metabolismo , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Endogâmicos/genética , Ratos Endogâmicos/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismo , Rodopsina/metabolismo , Transducina/genéticaRESUMO
Glutathione-insulin transhydrogenase (GIT, thiol:protein-disulfide isomerase/oxidoreductase, E.C. 5.3.4.1/1.8.4.2) catalyzes via sulfhydryldisulfide interchange, the scission as well as formation of disulfide bonds in many diverse proteins. Using insulin as a substrate, the lens epithelial layer of cows, rats and rabbits was found to contain GIT activity. The enzyme's activity is activated by GSH and inhibited by N-ethylmaleimide. Subcellular distribution of bovine lens epithelial homogenates showed that the majority of GIT activity is located in the insoluble fraction (10,000 g pellet) and in the high molecular weight fraction (60,000 g pellet). Lens epithelial extracts were subjected to SDS-PAGE followed by Western blot, and probed with a polyclonal antibody to rat liver GIT, or with either of two monoclonal antibodies directed against different epitopes of the enzyme. Lens epithelium was found to contain two forms of GIT, one with the same molecular weight as the purified enzyme (Mr 56Kd), and a second having an Mr of 67Kd. Immunoblots using polyclonal antibodies revealed an additional major immunoreactive band of 32Kd in the cow lens epithelial layer as well as in the isolated cortical and nuclear portions. Rat lenses showed no immunoreactive 32Kd band. Using a bovine cortical/nuclear fraction the 32Kd reactivity was found to be associated with the beta H-crystallin fraction, but the extract failed to show GIT activity with the insulin substrate. This suggests that beta H-crystallin may share a common epitope with GIT.
Assuntos
Cristalino/enzimologia , Oxirredutases/metabolismo , Proteína Dissulfeto Redutase (Glutationa)/metabolismo , Animais , Bovinos , Immunoblotting , Cápsula do Cristalino/enzimologia , Cápsula do Cristalino/ultraestrutura , Peso Molecular , Coelhos , Ratos , Ratos Endogâmicos , Frações Subcelulares/enzimologia , Distribuição TecidualRESUMO
Vitamin E (alpha-tocopherol) is the major lipid-soluble antioxidant of retinal membranes whose deficiency causes retinal degeneration. Its antioxidant function is realized via scavenging peroxyl radicals as a result of which phenoxyl radicals of alpha-tocopherol are formed. Our hypothesis is that alpha-tocopherol phenoxyl radicals can be reduced by endogenous reductants in the retina, providing for alpha-tocopherol recycling. The results of this study demonstrate for the first time that: (i) endogenous ascorbate (vitamin C) in retinal homogenates and in rod outer segments is able to protect endogenous alpha-tocopherol against oxidation induced by UV-irradiation by reducing the phenoxyl radical of alpha-tocopherol, (ii) in the absence of ascorbate, neither endogenous nor exogenously added glutathione (GSH) is efficient in protecting alpha-tocopherol against oxidation; (iii) GSH does not substantially enhance the protective effect of ascorbate against alpha-tocopherol oxidation; (iv) exogenous dihydrolipoic acid (DHLA), although inefficient in direct reduction of the alpha-tocopherol phenoxyl radical, is able to enhance the protective effect of ascorbate by regenerating it from dehydroascorbate. Thus, regeneration of alpha-tocopherol from its phenoxyl radical can enhance its antioxidant effectiveness in the retina. The recycling of alpha-tocopherol opens new avenues for pharmacological approaches to enhance antioxidants of the retina.
Assuntos
Ácido Ascórbico/fisiologia , Retina/metabolismo , Ácido Tióctico/análogos & derivados , Vitamina E/biossíntese , Animais , Cromatografia Líquida de Alta Pressão , Ácido Desidroascórbico/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Glutationa/farmacologia , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Retina/efeitos dos fármacos , Retina/efeitos da radiação , Ácido Tióctico/farmacologia , Raios UltravioletaRESUMO
PURPOSE: Clusterin has been associated with active cell death in several different model systems, including animal models of retinal degeneration. Clusterin is also expressed in normal tissues, a finding that leads to the question of how it could then play a cell death-specific role during tissue regression. To address this paradox, we have examined clusterin expression during light-induced retinal damage in rats. METHODS: Normal albino rats were reared in darkness and then exposed to intense visible light to induce retinal degeneration. Clusterin expression was then examined at various times after light treatment. Standard molecular techniques including Northern analysis, immunohistochemistry, and Western analysis were employed. RESULTS: Northern analysis established that the largest increase in clusterin expression occurs after a decrease in interphotoreceptor retinoid binding protein, IRBP, expression (an indication of a photoreceptor cell dysfunction) and after an increase in heme oxygenase 1, HO-1, expression (an oxidative stress inducible gene), suggesting that induction of clusterin expression is an oxidative stress response. Immuno-histochemical analysis with two different clusterin-specific antibodies, anti(SGP-2) and anti(301), localized distinct forms of clusterin to Müller cells and degenerating photo-receptor cells. Western analysis demonstrated degeneration associated isoforms of clusterin in light treated retina that are not present in normal retina. CONCLUSION: Clusterin over-expression is characteristic of a retinal degeneration phenotype and we propose that clusterin action may be defined by the nature in which it is modified. We hypothesize that alternate processing leads to retinal degeneration-specific forms of the protein (65, 61, and 50 kDa) that are not present in normal retina.
Assuntos
Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Chaperonas Moleculares/metabolismo , Lesões Experimentais por Radiação/metabolismo , Retina/efeitos da radiação , Degeneração Retiniana/metabolismo , Animais , Northern Blotting , Western Blotting , Clusterina , Adaptação à Escuridão , Proteínas do Olho/genética , Expressão Gênica , Glicoproteínas/genética , Heme Oxigenase (Desciclizante)/metabolismo , Heme Oxigenase-1 , Técnicas Imunoenzimáticas , Luz , Masculino , Chaperonas Moleculares/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/patologia , Ratos , Ratos Sprague-Dawley , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Proteínas de Ligação ao Retinol/metabolismoRESUMO
The effects on the uptake (cell-associated 125I) and degradation (125I-labelled products released into the medium) of 125I-insulin and bioactivity (protein, glycogen and lipid synthesis) of insulin caused by altering the cellular thiol/disulphide status in primary cultures of rat hepatocytes were studied. Incubation of hepatocyte cultures with various exogenous thiol compounds (reduced glutathione, 2-mercaptoethanol, cysteamine, dithiothreitol) resulted in increased insulin binding, but markedly decreased degradation and bioactivity. These effects could be reversed by washing or by the addition of oxidized glutathione, which alone had no effect. When cultures were exposed to certain thiol-modifying reagents (N-ethylmaleimide, p-chloromercuribenzoate, p-chloromercuribenzenesulphonate, iodoacetamide, iodoacetate), some decreases in bioactivity were evident, but the pronounced decrease in insulin degradation observed with the thiol-containing compounds was not observed with this class of compounds. None of the thiol-containing or -modifying agents tested had any significant effect on cellular ATP concentrations, indicating that the effects observed were due to perturbation of the thiol/disulphide status. Depletion of intracellular glutathione by DL-buthionine SR-sulphoximine (a specific inhibitor of glutathionine biosynthesis) decreased the syntheses of glycogen and lipid by about one-half, while having essentially no effect on protein synthesis, ATP concentrations or on the binding and degradation of insulin. The data presented here indicate that although intracellular thiol (glutathione) concentrations may be important for the maintenance of full expression of certain biological activities (glycogen and lipid synthesis), the thiol/disulphide groups on the cell surface and those immediately inside the cell membrane may be more critical in the mediation of insulin action, including the degradation and bioactivity of insulin in primary cultures of rat hepatocytes.
Assuntos
Dissulfetos/metabolismo , Insulina/metabolismo , Fígado/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Butionina Sulfoximina , Células Cultivadas , Ditiotreitol/farmacologia , Glutationa/farmacologia , Fígado/citologia , Masculino , Metionina Sulfoximina/análogos & derivados , Metionina Sulfoximina/farmacologia , Ratos , Ratos Endogâmicos , Compostos de Sulfidrila/farmacologia , Reagentes de Sulfidrila/farmacologiaRESUMO
Incubation of primary cultures of rat hepatocytes with the local anesthetics, procaine or lidocaine, had little or no effect on insulin uptake or degradation but caused an inhibition of insulin-stimulated glycogenesis. While exposure of cultures to the amines, monodansylcadaverine or CH3NH2, resulted in significant dose-dependent decreases in glycogenesis, only monodansylcadaverine (an inhibitor of receptor clustering) decreased uptake whereas CH3NH2 (a lysosomotropic agent) caused increases in both insulin uptake and degradation. When cells were treated with agents which inhibit glycolysis (NaF, 2-deoxy-D-glucose) or oxidative metabolism (2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl hydrazone, NaN3, antimycin A), pronounced inhibitions of each of the bioactivities studied (syntheses of glycogen, protein, lipid) were observed, but only the glycolytic inhibitors decreased insulin uptake. These results suggest that insulin is internalized by an endocytotic process involving receptor clustering and requiring metabolic energy derived from glycolysis. The post-receptor biosynthetic processes involved in the expression of the biological activities of insulin (syntheses of glycogen, protein, lipid) require energy produced by oxidative metabolism while the degradation of insulin is carried out by nonlysosomal mechanisms which are not energy-requiring.
Assuntos
Membrana Celular/metabolismo , Metabolismo Energético/efeitos dos fármacos , Insulina/metabolismo , Fígado/metabolismo , Aminas/farmacologia , Anestésicos/farmacologia , Animais , Antimetabólitos/farmacologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Transporte de Elétrons/efeitos dos fármacos , Endocitose , Glucose/biossíntese , Glicólise/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fosforilação Oxidativa/efeitos dos fármacos , RatosRESUMO
Previously, we described primary cultures of rat liver cells in a serum-free multihormone defined medium (Am. J. Physiol. 243: E132-E151, 1982). This cell preparation exhibits marked insulin-dependent syntheses of protein, glycogen and lipids (hereafter collectively referred to as "bioactivity" for brevity). In the present studies, the role of different hormones in the expression of insulin's bioactivity was investigated. Hepatocytes from fasted rats, previously maintained in the multihormone glucagon-supplemented medium, were first cultured in the defined medium without the hormones and, subsequently, tested for bioactivity by replacing the missing hormones singly or in combination. Of all the hormones tested (testosterone, estradiol, thyroxine, glucocorticoid steroid and insulin), only insulin, dexamethasone and thyroxine when present individually in the culture medium, showed slight bioactivity (glycogenesis); however, insulin and dexamethasone, when present together, further increased each bioactivity, protein (1.3-fold), glycogen (8-fold) and lipid (1.6-fold) syntheses. The determination of apparent binding parameters of insulin specific receptors using Scatchard plots showed that insulin exposure caused a decrease in receptor concentration (Ro), dexamethasone exposure caused an increase in affinity (Kd), and, compared to insulin alone, treatment with insulin plus dexamethasone increased receptor concentration with no change in apparent affinity. Insulin induced a consistently small, but statistically significant, increase in the average specific activity of the protein-disulfide interchange enzyme, glutathione-insulin transhydrogenase (GIT), with equal distribution between its nonlatent ("readily available") and latent ("cryptic") forms. However, when dexamethasone was present along with insulin, the distribution of GIT was significantly greater in the latent form than in the nonlatent form. Examination by scanning and transmission microscopy showed clear differences, both in the cell surface and intracellular morphology, in the hepatocytes exposed to different hormonal milieu.